Functionality of unliganded VDR in breast cancer cells: repressive action on CYP24 basal transcription

Abstract

It is well-established that CYP24, an immediate target gene of VDR is upregulated by VDR ligands. This study is focused on the functional role of unliganded VDR by investigating the correlation between the expression of VDR protein and basal mRNA levels of CYP24 in breast cancer cell lines. Analyses of multiple breast cancer cell lines demonstrated an inverse correlation between VDR protein expression and CYP24 mRNA expression levels; while in the presence of ligand, VDR protein level was positively correlated with CYP24 expression. In MCF-7 cells, VDR was mainly distributed in the nuclei in the absence of ligand. VDR overexpression in MCF-7 cells and MDA-MB231 cells decreased CYP24 mRNA expression levels and CYP24 promoter activity. Conversely, knock-down of VDR using siRNA techniques in MCF-7 and T47D cells significantly increased CYP24 mRNA expression. We also found that overexpression of VDR with a polymorphic site (FokI-FF) at its AF-1 domain, which makes VDR shorter by three amino acids, failed to repress CYP24 promoter activity. This report provides conclusive evidence for the repressive action of unliganded VDR on the expression of its target gene CYP24 and the importance of an intact VDR AF-1 domain for its repressive action.

Fig. 1

Differential regulation of CYP24 mRNA expression by VDR in the absence (a) and presence of ligand (b) and VDR nuclear localization in MCF-7 cells (c). a Protein levels are inversely correlated to CYP24 basal mRNA expression in the absence of the ligand. Upper panel: qRT–PCR analysis of CYP24 mRNA expression in different breast cancer cells. CYP24 basal mRNA levels in the BT474 samples were set as one after normalization to β-actin. CYP24 mRNA level in each sample was normalized to β-actin and the basal CYP24 level of BT474 cells. Duplicate real-time PCR analyses were carried out for each RT sample. The results are expressed as a mean ± SEM of three experiments. Lower panel: VDR protein expression in different breast cancer cell lines, β-actin served as a loading control. b VDR protein levels positively correlated with CYP24 induction in the presence of the ligand. Cell were treated with 0.5 μM 1α(OH)D₅ for 24 h and analyzed for CYP24 transcription by RT–PCR (upper panel) and VDR protein by western blot (lower panel). c Immunofluorescent staining of VDR in MCF-7 cells in the absence of ligand. Upper panel: nuclei stained with DAPI; lower panel: unliganded VDR (green) is localized in the nuclei of MCF-7 cells

Fig. 2

Effect of VDR overexpression on CYP24 expression and promoter activity in breast cancer cell lines. a MCF-7 cells were transiently transfected with VDR expression vector, CYP24 mRNA (upper panel) and VDR protein (lower panel) were analyzed at 48 h after transfection. b CYP24 basal transcriptional levels inversely correlated with VDR protein levels in MDA-MB231 cells stably transfected with VDR expression vector. All the results are expressed as a mean ± SEM of three samples obtained from three independent experiments. A two-tailed student’s t-test was performed to determine any significant differences between control and VDR-transfected cells (* P<0.05, ** P<0.01). c and d Overexpression of VDR repressed CYP24 promoter activity in both MCF-7 and MDA-MB231 cells MCF-7 (c) and MDA-MB231 cells (d) were co-transfected with VDR expression vectors or empty vector (pcDNA3.1) and CYP24 promoter–luciferase reporter construct. At 24 h after transfection, CYP24 promoter activity was evaluated by luciferase assay. Data are expressed as a mean ± SEM of three-independent experiments with at least triplicate wells for each transfection. * P<0.05 in comparison to control

Fig. 3

Decreased VDR protein expression was accompanied by increased CYP24 expression in breast cancer cells. MCF-7 (a) and T47D (b) cells were transfected with siVDR or si- Control. CYP24 mRNA expression (upper panel) and VDR protein level (lower panel) were examined 36 h after transfection. Results are expressed as a mean ± SEM of three samples obtained from two independent experiments. * P<0.05, ** P<0.01 in comparison to control

Fig. 4

Effect of VDR Fok1 polymorphism on CYP24 promoter activity and CYP24 mRNA expression. a and b MCF-7 and MDA-MB231 cells were co-transfected with VDR expression vectors (containing the FokI site, VDRff, and VDRFF, respectively) or empty vector (pcDNA3.1) and CYP24 promoter reporter construct. CYP24 promoter activity was evaluated 24 h after transfection. Data shown are representatives of two independent experiments. Bar, mean ± SEM of four samples; ** P<0.01, * P<0.05 in comparison to vector control. c Upper panel: CYP24 promoter activity in MCF-7 cells stably transfected with VDRff. Bar, mean ± SEM of three independent experiments, *** P<0.001, n = 3; lower panel: VDR expression in the MCF-7 cells expressing empty vector and VDRff. β-actin served as a loading control. d RT–PCR analysis of CYP24 expression of both unspliced and spliced forms. qRT–PCR analysis of β-actin demonstrated equal loading for the two lanes (data not shown). M maker, vector MCF-7 cells stably expressing empty vector, VDRff MCF-7 cells stably expressing VDR with an intact AF-1 domain